Phosphorylation of smooth muscle and nonmuscle myosin II is required to activate contraction. A single serine residue in the regulatory light chain of each of the two "head" domains of myosin is phosphorylated by a Ca2+-activated kinase. The current knowledge suggests that this dramatic regulatory effect is mediated through large conformational changes in the myosin structure. The long term goal of this proposal is to determine the structural basis of the phosphorylation-dependent regulatory mechanism. The P.I. has recently demonstrated that both of the two identical head domains of this myosin are required for phosphorylation-dependent regulation. Based upon these and other data a hypothesis is proposed, which is guided by the recent three-dimensional structural model of skeletal myosin and the regulatory domain of scallop myosin. Hypothesis: Interaction between the two myosin head domains is favored in the unphosphorylated "off" state, and the role of phosphorylation is to diminish these interactions, thus allowing for myosin to adopt force generating states in the presence of actin. The specific aims of the proposal are to test the hypothesis by answering two related questions: l) Does an interaction between the myosin heads occur under conditions predicted by the hypothesis? 2) Which specific elements of the myosin structure are involved in the putative head-head interaction? To accomplish the specific aims, fluorescence, photocrosslinking and iron- BDTA mediated peptide cleavage experiments will be performed under well- defined experimental conditions which are designed to test the hypothesis. In addition, the relationship between structure and activity of a novel new class of engineered nonmuscle myosins that contain one unmodified complete head and one partial head will be studied to specifically fulfill aim #2.